Laboratory procedures in biotechnology frequently include the detection, identification, and quantitation of individual proteins. Certain procedures also however require the quantitation of entire mixtures of proteins aside from the individual proteins in each mixture, and with or without separating or differentiating the individual proteins from each other. Such total protein quantitation is of value, for example, when comparing protein amounts from different samples, including samples from different subjects, different organisms, and different species, or from the same species or subjects at different stages of development or different stages of disease or disease conditions. Total protein determinations in these cases can reveal quantitative changes in gene expression, which can be of value of diagnosis, treatment, and the testing of therapeutic agents. Total protein determinations can also be used to check sample loadings and protein transfers in analytical systems. When loading samples into individual wells of a microplate or individual lanes of an electrophoresis gel, for example, a uniform and consistent loading among the various wells or lanes is needed to assure that proper comparisons can be made in any of the separations or other procedures that might be performed on the samples. Protein transfers arise in Western blots, for example, which are also an area of concern, since total protein quantitations can indicate whether a blot has been performed correctly or an incomplete transfer has occurred.
Protein detections and quantitations using conventional stains such as COOMASSIE™ Brilliant Blue (BASF Aktiengesellschaft, Ludwigshafen, Germany), Ponceau S (Sigma-Aldrich, St. Louis, Mo., USA), and SYPRO RUBY™ (Sigma-Aldrich) are well known. A stain-free technique is disclosed by Edwards et al. in U.S. Pat. No. 7,569,103 B2 (Aug. 4, 2009) and U.S. Pat. No. 8,007,646 B2 (Aug. 30, 2011), citing the UV light-induced reaction between the indole moiety of tryptophan and any of various halo-substituted organic compounds, with specific mention of chloroform, trichloroethanol, and trichloroacetic acid, to produce a fluorescent compound with emissions at wavelengths in the visible range. The technique thus entails treating the proteins, or the gels in which the proteins reside, with the halogen-containing reagent, exposing the proteins or gels to UV light, and detecting and quantifying the resulting emission for individual protein bands.
Tryptophan is relatively rare in proteins, and the proportion of tryptophan residues to total amino acid residues within each protein varies widely from one protein to the next. For this reason, recognition of the usefulness of the stain-free technique to date has been limited. The technique is thus known to be useful for comparing different samples of the same protein, for example, by comparing the intensity of a single band of the same molecular weight protein in each sample. Comparisons between different protein bands in different samples would not be useful, since the different proteins are most likely to have different tryptophan contents and thus cannot be expected to produce the same signal intensities when present in the same amounts. The tryptophan variances do not necessarily limit the usefulness of the technique to the comparison of individual proteins, however. Total protein determinations are valid when performed on samples from the same source. The technique can be used in a blotting experiment for example for comparing the stain-free signal in a gel before and after blotting to determine if any protein has remained in the gel. The technique can also be used for absolute quantitation by creating a standard curve using a range of known concentrations of a protein and evaluating a sample containing an unknown amount of the same protein provided that the amount of the protein the sample is within the range of the standard curve. Nevertheless, in all cases the comparisons are direct comparisons either between the same sample before and after some form of manipulation, or of the same individual protein in different samples. In neither of these cases would any differences in tryptophan contents between different proteins negate the validity of the result.
When investigating an individual protein of interest a sample, it is sometimes beneficial to normalize the amount of that protein with respect to one or more other proteins in the sample, or with respect to all proteins in the sample. Normalization is a means to correct experimental data to accommodate for differences in sample dilution, sample loading, or protein transfer inconsistencies. For example, if two different cell cultures are used as samples it is likely that each culture contains different numbers of cells. If each cell in both samples has the same amount of a particular protein then the culture with more cells will have more of the protein. Without knowing that one sample contained more cells than the other one would incorrectly conclude that an average cell in one culture had more of the protein than an average cell in the other culture. One uses normalization to correct for this type of difference.
The most common normalization method is the use of housekeeping proteins (HKPs). In order to be considered a HKP a protein must be present in approximately the same quantity in all samples. Tubulin, GAPDH, and actin are frequently chosen as HKPs due to their general lack of variability associated with changes in experimental conditions. As a number of publications have shown, however, caution should be used when selecting HKPs since not all HKPs are constant under changes in experimental conditions or sample types. For this reason, it is often recommended that results are validated using multiple HKPs, adding time and complexity to western blot experiments.
The process of using HKPs for blot normalization can be daunting. Two frequently used techniques are “strip and reprobe” and multiplex fluorescent detection. Regardless of the method used, HKP-based normalization with GAPDH, actin or tubulin needs to be optimized for linear range of detection, antibody dilutions, incubation times and imaging settings.
Using the strip and reprobe method, the protein of interest is probed and detected. Antibodies and detection chemistries are then stripped from the membrane using some combination of heat, detergent, or reducing agent. The blot may then be re-probed with HKP specific antibody and re-detected. Not only is the process time consuming, but inevitably, the stripping process will remove some level of antigen, thereby compromising downstream results.
Multiplex fluorescent western blotting is a more elegant solution, whereby multiple antigens can be simultaneously probed and detected using multiple fluorescently labeled secondary antibodies. Multiplex Western blotting requires the same optimizations as noted above, but has other challenges like antibody cross-reactivity, and should have an optimization process in place that validates the detection of each antigen separately, before attempting a multiplex detection.
If one wishes to avoid the challenges associated with the use of HKPs by stripping and reprobing, or with optimization of multiplex fluorescent blot detection, one can instead use a technique called total protein normalization (TPN). There are three commonly used methods for perform TPN. The first is to perform a protein assay on the samples prior to analysis and then to dilute them to the same concentration for the analysis. This is commonly done using a Bradford dye assay, a bicinchoninic acid (BCA) assay, or other similar methods. Unfortunately this method assumes that there is no variability within the process and this is not always the case. Any pipetting variation as samples are prepared and loaded onto the gel is not accounted for nor does this method account for variations in the blotting efficiency between the gel and the membrane. Another method of performing TPN is to stain the gel after it is run. This can be done using reversible stains such as zinc or copper based stains that bind to the proteins within the gel for detection yet can be washed away so as not to interfere with the blotting process and subsequent immunodetection. Although this method accounts for variations in the sample preparation and gel loading it does not account for blotting efficiency and it adds time and complexity to the process. The third method for performing TPN is to use colored or fluorescent stains on the membrane such as SYPRO Ruby, Ponceau S, and Amido Black. This method accounts for variability in sample preparation and transfer efficiency but it still adds time and complexity to the process.
The present invention provides a new and easier method, one that performs TPN by use of stain-free technology, thereby avoiding the difficulties associated with HKP and eliminating the drawbacks with existing TPN methods.